Production and performance of sugarcane seeds ( caryopses ) from different hybridizations involving RB 92579

A major challenge in sugarcane breeding program is the obtaining of enough number of seeds (caryopses) for the development of new improved cultivars. Genotypes differ in their function as pollen recipient and pollen donor, which also affect the seed performance. Thus, the aim of this study was to verify the production and performance of sugarcane seeds from different hybridizations involving RB92579 as pollen recipient and pollen donor. Twelve bi-parental crossings were carried out involving RB92579 and other different genotypes randomly chosen. Seed production potential was evaluated by percentage of fertile spikelets and caryopsis fresh weight. The seed physiological potential was determined by evaluating germination and vigor (index of germination rate, number of normal seedlings per gram of fuzz, and seedling dry weight). The results showed better performance for RB92579 as pollen donor for all characteristics studied. Therefore, RB92579 sugarcane cultivar should be used as pollen donor during hybridizations, condition that permits a greater production and physiological performance of seeds for the sugarcane breeding programs.


INTRODUCTION
Sugarcane (Saccharum spp.) is the world's most important sugar-producing crop.Global interest in sugarcane has also increased because of its economic impact on the sustainable energy production (Hoang et al. 2015).Sugarcane breeding programs have been working extensively to the development of hybrid seeds (caryopses) with desirable agroindustrial traits (Cheavegatti-Gianotto et al. 2011).The Northeastern region of Brazil is a traditional producer of sugarcane, and RB92579 cultivar is one of the major cultivar used in hybridization of sugarcane in this region.RB92579 is characterized by high productivity and adaptability to adverse environmental conditions that can be inherited by their descendants (Daros et al. 2015).
A major challenge in sugarcane breeding program is the obtaining of enough number of seeds for the development of new improved cultivars (Heinz and Tew 1987).Genotypes differ widely in their role as maternal parent (pollen recipient) or paternal parent (pollen donor).This can negatively affect seed production and later performance CARLOS A. DINIZ et al. (McIntyre and Jackson 2001).Accordingly, it is particularly important to determine the responses of each cultivar as pollen recipient and pollen donor during hybridization.The incorrect use of a genotype as pollen donor, for instance, can cause molecular or physiological changes that may affect seed quality and production potential.Therefore, differences between inflorescence condition as pollen recipient and pollen donor should always be considered during the hybrid seed production.
Moreover, since the seedling performance is directly dependent on seed vigor (Silva et al. 2012), higher seed vigor plays an important role during breeding programs because high-vigor seed lots are expected to produce strong seedlings with ability to express their real genetic traits.In addition, the use of high-vigor seed lots assures more uniform stand of plants in the field (Marcos-Filho 2015), which can also contribute more efficiently to the selection of agronomically desirable genotypes for the breeding programs.
Thus, this research aimed to study the production and performance of sugarcane seeds from different hybridizations involving RB92579 as pollen recipient and pollen donor.

MATERIALS AND METHODS
Crossing experiments of sugarcane cultivars were conducted at the Sugarcane Flowering and Crossing Station Serra do Ouro, located in Murici, AL, Brazil (9º13'S, 35º50'W, 450 m asl) from May to June 2014.Evaluations were carried out at the Plant Propagation Laboratory and in a greenhouse, located at the Center of Agricultural Sciences, Federal University of Alagoas, Rio Largo, AL, Brazil from October 2014 to April 2015.
The hybridization process was conducted using the method described by Barbosa et al. (2002).After seed processing, the seeds were kept in multi-layered Kraft paper containers and stored at 20 ºC and 50% relative air humidity during the experimental period.

SEED PRODUCTION POTENTIAL
Seed production potential was evaluated for the percentage of fertile spikelets and caryopsis fresh weight.The percentage of fertile spikelets was determined with three replicates of 0.25 g 'fuzz' (glumes, lemma, palea and caryopses), which was evaluated manually for the absence or presence of caryopsis.For caryopsis fresh weight, three replicates of caryopses were used for each crossing.All the caryopses were weighed, and the results were expressed as individual fresh weight of caryopsis for each crossing.

SEED PHYSIOLOGICAL POTENTIAL
Seed physiological potential was evaluated by germination and vigor tests (index of germination rate -GR, number of normal seedlings per gram of fuzz, and seedling dry weight).Seed germination tests were conducted with three replicates of 100 spikelets per crossing.Spikelets were distributed on paper towel moistened with water equivalent to 2.5 times the paper weight, and then placed at bottom of plastic boxes (11.0 x 11.0 x 3.5 cm) under alternating temperature of 20-30 ºC and continuous light (Caieiro et al. 2010).The number of normal seedlings was recorded daily to obtain index of germination rate (Maguire 1962).Germination percentage was calculated at 10 days after sowing (Silva et al. 2010); results were expressed as a percentage of normal seedlings (Brasil 2009).
(pollen recipient) has shown lower percentage of fertile spikelets, in the opposite condition, that is, RB92579 as pollen recipient and RB981809 as pollen donor, RB981809 showed the best performance (11.0%);RB925211 showed the worst fertile spikelets values (1.6%).
In this research, the highest fertility of sugarcane (50.2%) was relatively low compared to other grasses (Fairey 1993).Pierre et al. (2015) also found low fertility in other sugarcane cultivars with an average of 31.8%.Previous studies with RB92579 demonstrated that the fertility values did not exceed 25% (Cabral et al. 2011).According to Creste et al. (2010), sugarcane genotypes have a high level of heterozygosity that results in marked genetic variability among progenitors and can reduce seed production.RB92579 as pollen donor showed higher caryopsis fresh weight (Table II) compared to the pollen recipient condition, especially during hybridization involving RB855511 cultivar, which showed the highest germination percentage (Table II).The seed germination percentage (Table II) from crossings with RB92579 as pollen donor varied between 4 and 37%.On the other hand, the results indicated lower performance for RB92579 For seedling emergence, three replicates with 1.0 g of fuzz were placed on a mix of black soil, coconut fiber and filter cake (2:1:1) in plastic boxes (40 x 30 x 15 cm) arranged in a greenhouse.A micro-sprinkler irrigation system was applied to moisten substrate.The boxes were covered with transparent plastic sheet to avoid the water loss.Evaluations were made 21 days after sowing and the results expressed as number of emerged seedlings per gram of fuzz.
Twenty one days after seedling emergence, seedlings were transferred to plastic tubes.At 60 days after sowing, the third tube of each experimental parcel was selected to determine the seedling dry weight.For this, seedlings were placed in kraft paper bags and kept in oven with forced air circulation at 65 °C for 72 hours.

EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS
Data were submitted to analysis of variance using a model linear hierarchical classification (Montgomery 2001).In each RB92579 condition, the parents means were compared using Scott-Knott test (p ≤ 0.05).

RESULTS AND DISCUSSION
The seed production and physiological potential (Table I) were statistically different between crossings involving RB92579 as pollen recipient and pollen donor.As pollen donor, RB92579 showed higher performance compared to its pollen recipient condition.
Overall greater production of fertile spikelets (Table II) was verified during hybridization between RB92579 (pollen donor) and RB925211 (pollen recipient) with 50.2% of fertile spikelets, whereas RB981809 (pollen recipient) showed the worst performance with 6.0% of fertile spikelets.
On the other hand, although hybridization between RB92579 (pollen donor) and RB981809 as pollen recipient in which case the highest germination percentage was 8%.
Since caryopsis fresh weight of RB92579 as pollen recipient was lower than pollen donor condition, it seems reasonable to assume that there were no enough stored reserves in the endosperm for providing nutrients to support early seedling growth with RB92579 as pollen recipient.
Moreover, the endosperm contains hydrolysis enzymes to the starchy endosperm, and the embryo utilizes sugars released by starch degradation for its growth (Yan et al. 2014).
The index of germination rate (Table II) involving RB92579 as pollen donor and RB855511 as pollen recipient was also higher

2015).
The number of normal seedling per gram of fuzz (Table II) was higher during hybridization between RB92579 (pollen donor) and RB855511 (pollen recipient).For seedling dry weight (Table II), results indicated that the best performance was exhibited by RB925211 (pollen recipient).
Depending on the genotype, sugarcane hybridization showed different performance, however, the best results were obtained by RB92579 as pollen donor.This can also be verified by a digital image of seedling roots of different hybridizations (Figure 1); seedlings from hybridizations involving RB92579 as pollen donor exhibited a more vigorous root growth compared to those produced from RB92579 as pollen recipient.Thus, higher seed vigor such as those that are the result of crossings with RB92579 as pollen donor, produced strong seedlings with more roots compared to those from lower seed vigor (RB92579 as pollen recipient).In general, a reduced root growth can limit the contact of roots to the soil in which there were grown, and plants can show difficult to establish in the field during transplant.In addition, a reduced root length also affects the water absorption capacity and consequently the crop yield.In this research, if seedlings produced from crossings involving RB92579 as pollen recipient had been transplanted to the field, probably they would have more difficulties to establish in the soil as well as reduced yield potential.
Therefore, seeds raised from RB92579 as pollen recipient showed clearly lower performance compared to its performance as pollen donor, with low capacity for female parent.Since the seedlings are obtained from sugarcane seeds during the first selection stage of a sugarcane breeding, such information is essential because can permit a more efficient selection of superior cultivars and allow them to express their real genetic potential; in fact, the higher the seed quality, the faster the seedling emergence, with capacity to tolerate better the adverse environmental conditions in the field (Marcos-Filho 2015).
Lower performance of RB92579 as pollen recipient can in part be explained by fertility problems because of problems with the ovule development, pollen-pistil incompatibility, maternal factors such as the position of the inflorescence on the mother plant or the position of the seeds along the inflorescence, or embryos fail to develop successfully (Gutterman et al. 2000, Alarmelu and Shanthi 2011, Schroeder et al. 2012, Melloni et al. 2013).Thus, although sugarcane flowers are hermaphrodites, RB92579 showed better performance as pollen donor, with predominant male reproductive function, possibly due to the greater degree of anther opening and higher pollen fertility.
In conclusion, for breeding programs the RB92579 sugarcane cultivar should always be used as pollen donor during hybridizations, condition that permit a greater seed production and performance.
than other genotypes reaching values of 11.4; for RB92579 crossed with other genotypes the index of germination rate varied from 1.2 to 4.4, and RB92579 as pollen recipient the highest index value was 2.1.For sugarcane breeding, genotypes are selected against flowering, and the energy produced is mainly used to increase crop yield (Berding and Hurney 2005).This can negatively affect fertility of flowers, seed production and performance (Olaoyeand Adams 1996, Cabral et al. 2011, Pierre et al.

Figure 1 -
Figure 1 -Digital images of sugarcane seedling roots from different hybridizations among RB92579 (as pollen recipient -a and pollen donor -b) and other six sugarcane cultivars.

TABLE I Percentage of fertile spikelets (FS), caryopsis fresh weight (CFW), germination (G), index of germination rate (GR), number of normal seedlings per gram of fuzz (NS), and seedling dry weight (SDW) from hybridizations involving RB92579 sugarcane cultivar as pollen recipient and pollen donor.
*Comparison of means within each row (F test, p ≤ 0.05).CARLOS A. DINIZ et al.